The goal of this project is the development of antiviral drug combinations composed of FDA-approved host- targeted compounds as therapeutic regimens with enhanced potency, broad-spectrum activity and high barriers to resistance. Resistance to antiviral therapy is a major public health concern, often undermining the clinical utility of whole classes of drugs targeting specific viral components. In the cases of rapidly mutating viruses such as HIV, hepatitis C, and influenza, resistance to antivirals can occur rapidly as the result of treatment with single agents. We propose a new avenue for drug development that may yield a new generation of drugs with high barriers to resistance and broad spectrum activity by repurposing approved drugs that simultaneously target multiple host factors and pathways that are critical for virus replication. By focusing on FDA-approved drugs, this approach could dramatically reduce the timeline and costs of bringing a product to market. More importantly, because these drugs target known and well validated host factors, and because their safety and efficacy have been demonstrated through human use, the risk associated with drug development is also significantly reduced. We screened a library of approved drugs and successfully identified >40 host-directed drug hits which had modest and differential activity against influenza and yellow fever viruses as single agents, but whose potency was enhanced 5- to 20-fold when used in combination with other host-targeted compounds or with direct-acting antivirals. In this R21/33, we propose to advance a lead compound (ADS-324, an antifolate) against a panel of Category A, B, and C viruses, and to identify and develop a combination regimen of approved HTDs with broad spectrum antiviral activity with the following four aims: Aim 1 (R21). Determine the spectrum of activity for ADS-324 against Category A, B, and C viruses in cell based assays. Milestone: Demonstration that ADS-324 has activity as a single agent (EC50 < 30 M) against more than one Category A, B, or C virus. Aim 2 (R21). Identify combinations of approved host targeted drugs with broad spectrum antiviral activity in vitro. Milestone: Identification of three or more combinations of hot targeted drugs which are active (EC50 >5-fold below clinically achievable concentrations of each drug) against more than one virus. Aim 3 (R33). Determination of the pharmacokinetic parameters of lead candidates in the appropriate model species. Milestone: Determination of the pharmacokinetic parameters (Cmax, AUC0-24, AUC0-, t 1/2) for up to four drugs in two species, and the estimation of human equivalent doses (AUC0-24). Aim 4 (R33). Demonstration of in vivo efficacy against Category A, B, or C viruses and nomination of lead candidates for development. Milestone: Demonstration of in vivo efficacy (>50% survival at clinically relevant doses) against at least one virus. At the successful completion of this proposal, we will be poised to submit an Investigational New Drug Application for a host-targeted antiviral regimen composed of approved drugs that act synergistically against multiple viruses.